Method for increasing ETEC CS6 antigen presentation on cell surface and products obtainable thereof

ABSTRACT

A method for increase the presentation of ETEC CS6 antigen on a cell surface, comprising the step of contacting cells expressing said antigen with an aqueous solution comprising 0.6-2.2 percent phenol by weight, such that the presentation of said antigen is increased by at least 100%. A method for the manufacture of a killed whole cell vaccine for immunization against CS6-expressing ETEC. Cells and vaccines obtainable by the above methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/726,957, filed on Oct. 6, 2017, which is a continuation of U.S.patent application Ser. No. 15/009,645, filed on Jan. 28, 2016, whichissued as U.S. Pat. No. 9,790,257 on Oct. 17, 2017, which is adivisional of U.S. patent application Ser. No. 14/239,732, filed on Feb.19, 2014, which issued as U.S. Pat. No. 9,273,103 on Mar. 1, 2016, whichis the U.S. National Phase Application of PCT International ApplicationNumber PCT/EP2012/067598, filed on Sep. 10, 2012, designating the UnitedStates of America and published in the English language, which is anInternational Application of and claims the benefit of priority toSwedish Patent Application No. 1150821-5, filed on Sep. 12, 2011, andU.S. Provisional Application No. 61/533,405, filed on Sep. 12, 2011. Thedisclosures of the above-referenced applications are hereby expresslyincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-NOV001-001C2.txt, the date of creation of the ASCII text file isSep. 10, 2019, and the size of the ASCII text file is 1.2 KB.

FIELD

The present invention relates to methods useful in the preparation ofETEC CS6 antigen, in particular for the manufacture of vaccines, as wellas cells and vaccines obtainable through the method.

BACKGROUND

Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrialcolonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC)bacteria, which are the most common cause of diarrhea among infants andchildren in developing countries and in travelers to such areas.

Since immune protection against ETEC is mainly mediated by locallyproduced IgA antibodies in the gut, much effort is focused on thedevelopment of an oral CF-based vaccine. ETEC candidate vaccinesinducing anti-CF immune responses have been developed, e.g. in the formof a combined CF-ETEC+CTB oral vaccine that contained five killed ETECstrains expressing several of the most commonly encountered CFs, i.e.CFA/I, CS1, CS2, CS3, CS4, and CS5, together with recombinant choleratoxin B subunit (CTB, which is highly homologous to the B subunit ofETEC LT) (Levine M M, Giron J A, Noriega F. Fimbrial vaccines. In: P.Klemm editor. Fimbriae: adhesion, biogenics, genetics and vaccines, CRCPress, Boca Raton, Fla. 1994, p. 255-70; Svennerholm A-M, Tobias J.Vaccines against enterotoxigenic Escherichia coli. Expert Rev Vaccines2008; 7:795-804).

Previous work has described the preparation of candidate E. coli vaccinestrains expressing immunogenic amounts of fimbrial CF antigens such asCFA/I and CS2, which are retained after formalin treatment. However,attempts to generate E. coli expressing immunogenic amounts of CS6 andto preserve the immunological activity of the CS6 protein in a killedwhole-cell vaccine have failed until now. Here is described theconstruction of a recombinant non-toxigenic E. coli strain, with anon-antibiotic selection marker thyA, which expresses large amounts ofCS6 antigen on the bacterial surface, and show that phenol inactivationof the bacteria does not destroy the CS6 antigen properties. To thecontrary, it was unexpectedly found that phenol treatment significantlyincreased the amount of antigen presented on the cell surface. Thisincrease is very relevant, since the number of cells that can beincluded in an oral whole cell vaccine is a major limiting factor invaccine development, due to the fact that too large numbers of bacteriagiven orally give rise to adverse effects such as vomiting, especiallyin infant subjects. By increasing the amount of antigen presented percell, the amount of antigen(s) can be increased in the vaccine withoutincreasing the overall number of cells in the vaccine.

Oral immunization of mice with such phenol-killed CS6 over-expressing E.coli bacteria induced strong fecal and intestinal IgA and serum IgG+IgMantibody responses to CS6 that exceeded the responses induced by an ETECreference strain naturally expressing CS6 and previously used as avaccine strain. The data indicate that the described phenol-inactivatednon-toxigenic and CS6 over-expressing E. coli strain is a usefulcomponent in an oral ETEC vaccine.

Definitions

In the context of the present disclosure, the terms below have thefollowing meanings.

The abbreviation ETEC refers to enterotoxigenic Escherichia colibacteria.

The term CS6 antigen means Coli surface antigen 6, one of the mostprevalent non-fimbrial colonization factors of ETEC bacteria. The termETEC CS6 antigen is used synonymously.

The term killed whole cell vaccine refers to vaccine containing whole(intact) but killed (non-living) bacteria.

The term non-antibiotic selection marker refers to genetic selectionmarkers for selection of plasmids not requiring the use of antibioticsin the selection process. Examples include thyA (thymidylate synthase)complementation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Construction of pJT-056-thyA for expression of CS6. Theplasmid pJT-CFA/I-ThyA was first constructed (FIG. 1A), and then theCFA/I operon was replaced with the entire amplified CS6 operon creatingpJT-056-thyA (FIG. 1B).

FIGS. 2A and 2B: Surface expression of CS6 on the C600-CS6 recombinantstrain and the CS6 reference strain E11881/23 examined by dot blot (FIG.2A) and inhibition ELISA (FIG. 2B). Both strains were cultured in liquidCFA medium, the recombinant strain induced with IPTG, both washed andtested in serial dilutions at an initial density of 109 bacteria/ml.**P<0.01 by Student's t test, two-tailed.

FIG. 3: Serum IgG+IgM, and fecal- and intestinal-extract ELISA IgAtiters against CS6, after oral immunization of C57 Bl/6 mice with thesame numbers of phenol-killed C600-CS6 bacteria and E11881/23 bacteria(n=5 mice/group). Titers are shown as geometric mean (GM)+standard error(SE) for the mice in each group; for fecal and intestinal extracts thelevels are adjusted to the total IgA levels in the extracts. Controlsrefer to antibody levels in mice before immunization. **P<0.01,***P<0.001, by Student's t test, two-tailed.

SUMMARY

In a first aspect, there is disclosed a method for increasing thepresentation of ETEC CS6 antigen on cell surface, comprising the step ofcontacting cells expressing said antigen with an aqueous solutioncomprising 0.6-2.2 percent phenol by weight, such that the presentationof said antigen is increased by at least 100%, preferably at least 200%,more preferably at least 300%.

Preferably, the duration of the contacting step is 1 to 72 h. Morepreferably, the duration of the contacting step is 1.5 to 42 h.

Preferably, the temperature during the contacting step is 18 to 42° C.,more preferably 18 to 38° C., even more preferably 18-22° C. or 36-38°C.

Preferably, the phenol concentration is 0.8-2.0 percent by weight, morepreferably 1.0-2.0 percent by weight.

Preferably, the cells comprise Escherichia coli cells. Preferably, theantigen is recombinantly overexpressed by the cells.

Preferably, the method of the first aspect further comprises the step ofcomparing the presentation of the antigen by the cells to thepresentation of CS6-antigen by untreated but otherwise comparable cellsby means of an inhibition ELISA or dot blot, preferably inhibitionELISA.

In a second aspect, there is also disclosed a method for the manufactureof a killed whole cell vaccine for immunization against CS6-expressingETEC, comprising the method according to any of the preceding claims,wherein the phenol concentration, the contacting temperature and thecontacting time are chosen such that at least 107-fold inactivation ofthe cells occurs concomitantly with the increase in CS6 antigenpresentation.

Preferably, in the method of the second aspect, the phenol concentrationis 0.6-2.0 percent by weight, the contacting time is 6-72 hand thecontacting temperature is 18-22° C. More preferably, the phenolconcentration is 0.75-0.85 percent by weight, the contacting time is40±2 hand the contacting temperature is 20±1° C.

In a third aspect, there is disclosed a cell obtainable by the methodaccording to the first or the second aspects.

In a fourth aspect, there is disclosed a vaccine for immunizationagainst CS6-expressing ETEC, comprising cells of the third aspect.

DETAILED DESCRIPTION

Method for Increasing Presentation of CS6-Antigen

In a first aspect, the present invention provides a method forincreasing the presentation of ETEC CS6 antigen on a cell surface,characterized by that it comprises the step of contacting a cell orcells expressing said antigen with an aqueous solution comprising0.6-2.2 percent phenol by weight, at a suitable temperature and for asuitable time, such that the presentation of said antigen is increasedby at least 20%. By the at least 20% increase in antigen presentation ismeant that the amount of antigen present of the cell surface anddetectable by suitable methods (see below for details) is at leastdoubled compared to cells not having been subjected to the method butwhich are otherwise comparable. Preferably, the increase in antigenpresentation is at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, morepreferably at least 100%, 125%, 150%, 175%, 200%, 250% or 300%. Mostpreferably, the increase is at least 100%.

The aqueous solution may e.g. be phosphate buffered saline (PBS) withadded phenol, but many different aqueous buffers are suitable. It ispreferable that the pH of the buffers is 5-9, more preferably 6-8, mostpreferably 6.5 to 7.5. The salt concentration of the buffer ispreferably 50-200 mM, more preferably 100-150 mM and most preferablyabout 137 mM. A suitable PBS buffer can be as follows: 8 g NaCl, 0.2 gKCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, in 1 liter, pH 7.4.

The variables phenol concentration, temperature and time exhibit acertain degree of interdependency. If the temperature is increased,lower phenol concentration and/or shorter time is required to achievethe increased presentation (and vice versa). If the treatment time islengthened, lower phenol concentration and/or lower temperature can beutilized (and vice versa). Aided by the guidance from the teachingsherein, the skilled person will be able to use routine experimentationwithout significant burden to adapt the combination of phenolconcentration, time and temperature to the needs at hand.

Suitable duration for the treatment may be 0.1 to 240 h or 1 to 240 h.Preferably, the treatment time may be 1 to 72 h. More preferably, thetreatment time may be 1.5 to 42.0 h. Most preferably, the treatment timeis 2.0-40.0 h.

Suitable temperature for the treatment is in the range of 1-45° C. or4-45° C. Preferably, the temperature may be 18 to 42° C. From practicalpoint of view, it may be preferable to perform the method at ambient(room) temperature to avoid the need for specialized equipment tomaintain the temperature. Thus one preferred temperature range is 18-25°C., even more preferably 18-22° C., most preferably about 20° C. It mayalso be preferable to perform the method at an elevated temperature toshorten the process duration and/or to reduce the required phenolconcentration. Thus another preferred temperature range is 35-42° C.,even more preferably 36-38° C., most preferably about 37° C.

The phenol concentration may preferably be in the range of 0.7 to 2.0percent by weight, more preferably 0.75-2.0 percent by weight, yet morepreferably 0.8-2.0 percent by weight, still more preferably 0.85-2.0percent by weight, even more preferably 0.9-2.0 percent by weight, andmost preferably 1.0-2.0 percent by weight. In one preferred embodiment,the phenol concentration is 0.6-2.0 percent by weight, the contactingtime is 6-72 h and the contacting temperature is 18-22° C. In anotherpreferred embodiment, the phenol concentration is 0.6-2.0 percent byweight, the contacting time is 2-4 h and the contacting temperature is36-38° C.

The cells in the above method may be Escherichia coli cells. This may beadvantageous from practical point of view since E. coli is readily grownand genetically manipulated in the laboratory. Furthermore, theprincipal purpose of the method is to provide vaccines against ETEC,whereby it may be advantageous to use an E. coli host for the antigen toprovide a more natural context for the antigen being presented.Preferably, the cells are non-toxigenic E. coli cells. Nevertheless, itshould be understood the method can be used with other CS6-expressingcells as well.

The cell concentration is not crucial within reasonable limits. Anyconcentration up to 10¹² cells/ml is considered feasible. A practicalpreferable cell concentration range may be 10⁸-10¹² cells/ml. The mostpreferable range is 10⁹ to 2·10¹⁰ cells/ml.

The ETEC CS6 antigen production may be recombinantly induced in thecell, by way of genetic engineering (see Example 1). This facilitatesthe production of high levels of the antigen per cell, advantageous forminimizing the number of cells needed for a vaccine dose, leading tominimized adverse effects. Nevertheless, the method can also be usedwith cells that natively (i.e. without any genetic engineering) expressthe CS6 antigen. Preferably however, the cells of the method arenon-toxigenic E. coli host cells that overexpress CS6-antigen as aresult of transformation with a CS6-expressing plasmid. Preferably, theplasmid has a non-antibiotic selection marker, i.e. a selection markerthat does not require the use of antibiotics for its function. Mostpreferably, the host cells are auxotrophic for thymidine and theCS6-overexpressing plasmid carriers a thymidylate synthase (ThyA)complementating factor, whereby the selection can be carried out using amedium devoid of thymidine. Preferably the CS6-overexpression is drivenby a tac-promoter or a similar strong inducible promoter well known inthe art.

In terms of measuring the increased presentation of the CS6 antigen as aresult of the method of the invention, useful methods are disclosedherein and are also known from the literature. Preferably, thedetermination is performed using an inhibition ELISA assay as disclosedherein (see Example 2 and the associated Materials and Methods), or bymeans of a dot blot also disclosed herein (see Example 2). Preferably,the method of the first aspect comprises the further steps of analyzingthe amount of presentation of the CS6-antigen by the cells (e.g. usingthe above techniques) and comparing the amount presented to the amountof CS6-antigen presented by cells which were not subjected to thetreatment with aqueous solution comprising phenol but which areotherwise comparable. Suitably, a portion of cells is taken aside andstored before the contacting step to serve as such a control sample.

Method for Manufacture of a Vaccine

In a second aspect, the present disclosure provides a method for themanufacture of a killed whole cell vaccine for immunization againstCS6-expressing ETEC, comprising the method according to the firstaspect, wherein the phenol concentration, contacting temperature andcontacting time are chosen such that at least 10⁷-fold inactivation ofthe cells occurs concomitantly with the increase in CS6 antigenpresentation. Preferably, the degree of inactivation is at least10⁸-fold, more preferably 10⁹-fold, yet more preferably 10¹⁰-fold andmost preferably there are no viable cells present after the treatment.Cell inactivation can be determined by any common means well known inthe art. A suitable method is disclosed herein in the section titledMaterials and Methods.

The utilization of phenol for cell inactivation as well as increasingantigen presentation may be advantageous since this reduces the numberof process steps in vaccine manufacture. The phenol inactivation alsosolves the problem resulting from the propensity of the CS6-antigenbeing destroyed the normally preferable inactivation method, formalintreatment (see Example 2).

Preferably, in the method of the second aspect, the phenol concentrationis 0.6-2.0 percent by weight, the contacting time is 6-72 h and thecontacting temperature is 18-22° C. Alternative preferred set ofconditions is where the phenol concentration is 0.6-2.0 percent byweight, the contacting time is 2-4 h and the contacting temperature is36-38° C. Yet another preferred set of conditions is where the phenolconcentration is 1.1-1.3 percent by weight, the contacting time is 16±3h and the contacting temperature is 20±2° C. Still another preferred setof conditions is where the phenol concentration is 1.1-1.3 percent byweight, the contacting time is 40±8 h and the contacting temperature is20±2° C. Further preferred set of conditions is where the phenolconcentration is 1.4-1.6 percent by weight, the contacting time is 6±2 hand the contacting temperature is 20±2° C. Yet further preferred set ofconditions is where the phenol concentration is 0.75-0.85 percent byweight, the contacting time is 40±2 h and the contacting temperature is20±1° C.

Cells and Vaccines Obtainable Through the Method of the Invention

In a third aspect, a cell obtainable by the method according the firstor second aspects is provided. The phenol treatment results in anapparent change in the structure of the cell wall and/or the antigensuch that more of the antigen is more available for detection (both invitro e.g. by antibodies and in vivo by the immune system). In otherwords, the bacterial cell thus treated has acquired a novel structure byway of the method of the first or second aspects, although it is notfeasible to describe the change in structure in structural terms.

In a fourth aspect there is provided a vaccine for immunization againstCS6-expressing ETEC, comprising cells according to the third aspect.

EXAMPLES

For details on the experimental procedures relating to the examples, thereader is referred to the section titled Materials and Methods.

Example 1: Expression of CS6 in E. coli C600-CS6

A DNA fragment carrying the structural genes (cssA, cssB, cssC, cssD)for CS6, prepared from a wild-type ETEC strain with surface expressionof CS6, was amplified by PCR and cloned to construct the expressionvector pJT-CS6-ThyA, as depicted in FIGS. 1A and 1B. This plasmid wasthen electroporated into the thymine dependent, non-toxigenic E. coliC600-ΔthyA strain, and CS6 surface expression was induced by addition ofIPTG to the growth medium, as shown in an immuno-dot blot assay (FIG.2A). No CS6 expression was observed in the absence of the inducer (datanot shown). When examining the expression of CS6 by the recombinantC600-CS6 strain using the dot blot assay, we found that this strainexpressed at least 8-fold higher levels of CS6 compared to the CS6reference strain E11881/23, which had previously been used as CS4+CS6vaccine strain in the CF-CTB-ETEC vaccine (FIG. 2A). Likewise, also whenspecifically determining the surface expression of CS6 using aninhibition ELISA assay, an approximately 10-fold larger amount of CS6was found on the recombinant strain as compared with the referencestrain (FIG. 2B).

Example 2: Inactivation of Bacteria without Destroying the CS6 AntigenProperties

With the aim to kill the CS6 expressing bacteria while preserving theCS6 antigen properties on their surface, the effects of formaldehyde andphenol were compared. Preliminary studies showed that treating thebacteria with 0.3% or 0.6% formaldehyde, while safely killing thebacteria, resulted in a complete loss of detectable CS6 antigen (datanot shown). In contrast, treating the bacteria with 0.5% phenol not onlykilled the tested bacteria, but also preserved the CS6 antigen (data notshown); a lower tested concentration of phenol, 0.25%, on the other handdid not result in complete killing of the bacteria. These resultsindicated that phenol treatment could be useful for inactivating thebacteria while preserving the CS6 antigen. To work out an optimalinactivation method, different concentrations of phenol were thereforetested for inactivation of both the recombinant C600-CS6 strain and forcomparison another CS6 over-expressing strain (TOP10-CS6-Amp). As seenin Table 2, with both strains tested with 0.5%, 0.8%, 1%, and 1.6%, butnot with 0.25%, of phenol inactivated the bacteria and also preservedthe surface CS6, as tested by inhibition ELISA. The maximal level of CS6was found when the bacteria were inactivated with 0.8% phenol, whichtreatment did in fact reproducibly increase the estimated amounts of CS6antigen surface compared to the untreated bacteria (Table 2). Based onthese results, phenol at the concentration of 0.8% was therefore used toinactivate both C600-CS6 and the reference strain E11881/23, whichresulted in killed bacteria with 6-fold larger amounts of CS6 on therecombinant strain than on the reference strain, as tested by inhibitionELISA. These inactivated bacteria were then used for oral immunizationsof mice.

TABLE 2 Surface CS6 levels and lack of growth of C600-CS6 andTOP10-CS6-Amp strains, after inactivation with difference concentrationsof phenol. CS6 (μg/10⁹ bacteria)^(a) Growth^(b) Phenol C600-CS6TOP10-CS6-Amp C600-CS6 TOP10-CS6-Amp 0 0.61 ± 0.038 0.39 ± 0.054 + +0.25%  0.55 ± 0.037 0.30 ± 0.06 + + 0.5% 0.53 ± 0.046 0.77 ± 0.084 − −0.8% 2.03 ± 0.23 2.36 ± 0.206 − − 1.0% 1.92 ± 0.18 1.87 ± 0.13 − − 1.6%1.15 ± 0.11 0.89 ± 0.082 − − ^(a)Levels of surface CS6 were measured byinhibition ELISA as described in materials and methods; values mean ± SEof four determinations. ^(b)Following inactivation with phenol, thetreated bacteria were tested for sterility (i.e. lack of growth) asdescribed in materials and methods; − indicates no growth, and +indicates growth.

Example 3: Immunogenicity of Phenol-Killed C600-CS6 in Mice

In a first test of the immunogenicity of the recombinant strain C600-CS6we immunized groups of both Balb/C and C57 Bl/6 mice with the samenumber of phenol-killed bacteria, and the serum antibody responses toCS6, as measured by ELISA, were compared. Although significant anti-CS6responses were induced in both types of mice, the antibody responses inC57 Bl/6 mice were substantially higher than in the Balb/C mice (datanot shown). We therefore used C57 Bl/6 mice for the further oralimmunization studies in which the immunogenicity of orally administeredphenol-killed vaccine preparation of C600-CS6 and the reference strainE11881/23 were compared. The results showed that all immunized miceresponded with production of serum IgG+IgM antibodies against CS6, andthat the titers of antibodies against CS6 on average were more than60-fold higher in mice immunized with C600-CS6 bacteria than those inmice immunized with the reference strain E11881/23 (FIG. 3) Fecal andintestinal IgA antibody responses against CS6 were also examined (FIG.3). In both cases the recombinant C600-CS6 strain induced significantly,on average 75-fold higher levels of fecal and intestinal IgA antibodiesagainst CS6 when compared to the levels in mice immunized with thecorresponding reference strain; the latter strain only inducedmarginally higher mucosal IgA anti-CS6 levels than those seen inunimmunized control mice.

Example 4: Optimization of Phenol Treatment Increasing CS6Antigen-Presentation at 20° C.

The C600-CS6 strain was cultured for overnight (16-18 h) in a rotaryshaker (150 rpm) at 37° C. Aliquots of the overnight culture was diluted1/100 and the resulting culture incubated for 2 h as above, after whichIPTG was added to a final concentration of 1 mM to induce expression ofCS6. The culture was then further incubated at the same conditions foran additional 6 h. The bacteria were then harvested, washed twice withPBS (to avoid presence of any residues from the medium), andre-suspended in PBS to a density of OD600=16 (corresponding toapproximately 2×10¹⁰ bacteria/ml).

The induced, OD-adjusted bacterial culture was divided into 8 flasks of250 ml, each containing 25 ml of the bacterial culture. As time zero(i.e. non-treated bacteria) a portion was taken for viable counting.Twenty five (25) ml of phenol to the final concentration (percent byweight) of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5, or 2.0 were added into theflasks, followed by incubation of all the flasks for 1 h, 2 h, 6 h, 16h, and 40 h, at room temperature with 80 rpm. After each time point, 1ml of each bacterial suspension (with phenol) was removed from eachflask to an Eppendorf tube. The bacteria were harvested, washedthoroughly twice with PBS (to avoid presence of any residues from thephenol), and re-suspended with 1 ml of PBS. The suspensions were storedat 4° C. and used for inhibition ELISA. The results are shown in Table3.

TABLE 3 Surface CS6 levels on C600-CS6 strain, after inactivation withdifferent concentrations of phenol for different times at 20° C. CS6(μg/10⁹ bacteria)^(a) Incubation times Phenol 0 h 6 h 16 h 40 h 0 0.30.6% 0.17 0.28 0.73 0.8% 0.28 0.77 0.89 1.0% 1.1 0.93 1.06 1.2% 1.67 2.21.36 1.5% 2.75 1.71 1.14 2.0% 1.57 0.91 0.37 ^(a)Levels of surface CS6were measured by inhibition ELISA as described in materials and methods.Values are the mean of two determinations

Example 5: Phenol Treatment Increasing CS6 Antigen-Presentation andSimultaneously Inactivating Bacteria in Industrial Scale

A 500 liter fermentor was inoculated with an E. coli strainoverexpressing the CS6 antigen (ETEX 24). After induction of expressionby IPTG the fermentation was continued for 8 hours. The bacteria wereharvested and washed over a 500 kD ultrafilter and finally dispensed ata concentration of 20×10⁹ bacteria/ml. Phenol was added to a finalconcentration of 0.8% (w/v) and the suspension was kept at 20° C. for 40hours under constant stirring. The suspension was washed over a 500 kDultrafiltration membrane in phosphate buffered saline and stored at 4°C.

During the inactivation procedure samples were taken before inactivationafter 1, 2, 18 and 40 hours of inactivation to test for viability.Briefly, samples taken were washed by centrifugation and resuspended inthe original volume in PBS whereafter dilutions were made in PBS andplated on Colonization Factor Agar (CFA agar). Plates were incubated at37° C. and counted the following day.

Inhibition ELISA to quantitate the amount of CS6 antigen was done onfresh material before inactivation and washed inactivated material.

It is clearly apparent that efficient cell inactivation and increasedCS6 antigen presentation can be achieved simultaneously in industrialproduction scale (Table 4).

TABLE 4 Time course for inactivation at 20° C. Inactivation time (h) 0 12 18 40 Total CS6 2.51 Nd Nd Nd 5.36 (μg/10⁹ cells) Bound CS6 1.01 Nd NdNd 2.90 (μg/10⁹ cells) Viable cells (cfu/ml) 1.64 × 10¹⁰ 1.5 × 10⁵ 1.2 ×10² 0 0 Nd = not determinedMaterials and Methods

Bacterial Strains and Culture.

The bacterial strains used in this study are listed in Table 1. Anon-toxigenic C600-ΔthyA E. coli strain, which is auxotrophic to thymine(N. I. A. Carlin and M. Lebens, unpublished) was used for constructionof the vaccine candidate strain C600-CS6. The ETEC strain E11881/23,which had previously been used as a CS4+CS6 expressing strain in theCF-ETEC+CTB vaccine, was used as a reference strain. For expression ofCS6, bacteria were grown in CFA medium (Evans D G, Evans D J Jr., CleggS, Pauley J A. Purification and characterization of the CFA/I antigen ofenterotoxigenic Escherichia coli. Infect Immun 1979; 25:738-48),supplemented with ampicillin (100 μg/ml) when necessary.

TABLE 1 List of strains, plasmids, and primers used in this studyStrains, plasmid and primers Relevant characteristic Reference/sourceStrains: ETEC GB35 CS6+, LT+ Nicklasson Microb Pathog, 2008;E. coli E11881/23 CS4+, CS6+, ST− 44:246-54. TOP10-CS6-AmpTOP10 expressing CS6, Amp^(r) Toblas Vaccine 2008; 26:5373-80E. coli C600-ΔthyA Auxotrophic to thymine; Kan^(r)NIA Carlin and M Lebens C600-CFA/I C600-ΔthyA/pJT-CFA/I-ThyA This studyC600-/CS6 C600-ΔthyA/pJT-CS6-ThyA This study Plasmid: pJT-CFA/I-Cm9041 bp Tobias Vaccine 2010; 28:6977-84. pNC-4 5086 bp; thyA NIA CarlinpJT-CFA/I-thyA 8879 bp; thyA This study pJT-CS6-ThyA 7973 bp; thyAThis study Primers: Primers: P15′-CGGTCTCCCTAGGCCTCCTTACCTATGGTGATC (SEQ ID NO: 1) P25′-CGGTCTCCTCGAGCGACTCTAGACCTAACCG (SEQ ID NO: 2) P35′-CGGTCTCGAATTCTAATGGTGTTATATGAAGAAAACAATTG (SEQ ID NO: 3) P45′-CGGTCTCAAGCTTAACATTGTTTATTTACAACAGATAATTGTTTG (SEQ ID NO: 4)

Construction of Expression Vector pJT-CS6-ThyA.

For construction of the C600-CS6 recombinant strain, the plasmidpJT-CFA/I-ThyA was first generated. The plasmid pJT-CFA/I-Cm (Tobias J,Holmgren J, Hellman M, Nygren E, Lebens M, Svennerholm A-M.Over-expression of major colonization factors of enterotoxigenicEscherichia coli, alone or together, on non-toxigenic E. coli bacteria.Vaccine 2010; 28:6977-84) was digested with XhoI and AvrII to remove thechloramphenicol resistance gene (cat). The plasmid pNC-4 (N. I. A.Carlin; unpublished) was then used in a PCR reaction to amplify the thyAgene (from Vibrio cholerae). The forward primer P1 (SEQ ID NO: 1) washomologous to a sequence starting 98 bp upstream of thyA and hadrestriction sites for Eco31I and AvrII, and the reverse primer P2 (SEQID NO: 2) was homologous to a sequence ending 75 bp downstream of thyA,and had restriction sites for Eco31I and XhoI, at the 5′ end (Table 1).PCR conditions were as follows: 95° C. for 5 min, 31 cycles of 94° C.for 15 s, 58° C. for 30 s and 72° C. for 50 sec, with a final extensionof 7 min at 72° C. The resulting 1065 bp fragment containing thyA wasthen gel-extracted and cleaved with XhoI and AvrII. Ligation of theamplified and digested thyA with the digested pJT-CFA/I-Cm resulted inpJT-CFA/I-ThyA (FIG. 1A). This plasmid was then electroporated into E.coli C600-ΔthyA, and a recombinant strain (C600-ΔthyA/pJT-CFA/I-ThyA)was isolated.

The plasmid pJT-056-ThyA was then constructed in two steps (FIG. 1B).First, the pJT-CFA/I-ThyA plasmid was digested with EcoRI and HindIII.PCR was used to amplify the CS6 operon from the CS6-expressing ETECstrain GB35 (Nicklasson M, Sjoling A, Lebens M, Tobias J, Janzon A,Brive L, Svennerholm A-M. Mutations in the periplasmic chaperone leadingto loss of surface expression of the colonization factor CS6 inenterotoxigenic Escherichia coli (ETEC) clinical isolates. MicrobPathog. 2008; 44:246-54). Amplification was carried out using theforward and reverse primers P3 (SEQ ID NO: 3) and P4 (SEQ ID NO: 4),respectively (Table 1) and the Expand High Fidelity PCR System (RocheDiagnostics GmbH). P3 is homologous to a sequence starting 13 bpupstream of cssA and carries restriction sites for EcoRI and Eco31I,whereas P4, which is homologous to a sequence ending 2 bp downstream ofthe cssD, carries restriction sites for HindIII and Eco31I, at the 5′end (FIG. 1B). PCR conditions were as described previously (Tobias J,Lebens M, Källgård S, Nicklasson M, Svennerholm A-M. Role of differentgenes in the CS6 operon for surface expression of enterotoxigenicEscherichia coli colonization factor CS6. Vaccine 2008; 26:5373-80). Theamplified CS6 operon, 4135 bp, was then restricted with Eco31I,resulting in a fragment with flanking EcoRI and HindIII, which wasligated with the digested pJT-CFA/I-ThyA resulting in a 7973 bppJT-056-ThyA plasmid. The constructed plasmid pJT-056-ThyA waselectroporated into the C600-ΔthyA strain. The resulting colonies werescreened for the presence of CS6 operon by PCR using the primers P3 andP4. Positive clones were further analyzed by restriction analysis ofisolated plasmids, and also by the ability to grow in CFA mediumconfirming their thymine independence. One such clone was selected as aCS6 positive and thymine independent strain, and designated C600-CS6(i.e. C600-ΔthyA/pJT-056-ThyA).

Expression of CS6.

The CS6 expressing strains were cultured in CFA medium, overnight (16-18h) in a rotary shaker (150 rpm) at 37° C. Aliquots of the overnightcultures were diluted 1/100 in CFA medium and the resulting cultureswere incubated for 2 h as above. Into the culture of the recombinantstrain, IPTG was added to a final concentration of 1 mM to induceexpression of CS6, and then cultures were further incubated at the sameconditions for an additional 6 h. The bacteria were then harvested andre-suspended in PBS to a density of OD600=0.8 (corresponding toapproximately 10⁹ bacteria/ml).

Quantification of CS6 on the Recombinant Strain.

A specific monoclonal antibody (MAb 2a:14) against CS6 (Helander A,Grewal H M, Gaastra W, Svennerholm A-M. Detection and characterizationof the coli surface antigen 6 of enterotoxigenic Escherichia colistrains by using monoclonal antibodies. J Clin Microbiol 1997;35:867-72) was used to quantify the level of expression of CS6 by therecombinant C600-CS6 and the ETEC reference E11881/23 strains, usingdot-blot and inhibition ELISA methods, as described (Tobias J, HolmgrenJ, Hellman M, Nygren E, Lebens M, Svennerholm A-M. Over-expression ofmajor colonization factors of enterotoxigenic Escherichia coli, alone ortogether, on non-toxigenic E. coli bacteria. Vaccine 2010; 28:6977-84and Tobias J, Lebens M, Bolin I, Wiklund G, Svennerholm A-M.Construction of non-toxic Escherichia coli and Vibrio cholerae strainsexpressing high and immunogenic levels of enterotoxigenic E. colicolonization factor I fimbriae. Vaccine 2008; 26:743-52.)

Preparation of Inactivated Bacteria.

Formaldehyde and phenol were tested and compared for inactivation of theCS6 expressing strains. Formaldehyde, in final concentrations of 0.3%(w/v; 0.1M) or 0.6% (0.2M), and phenol in final concentrations of0.25%-1.6% (w/v; 0.026-0.17M), were added to bacterial cultures at adensity of 10¹⁰ bacteria/ml in PBS. The suspensions were incubated for 2h at 37° C. with shaking at 60 rpm, and then kept at 4° C. for 3 dayswithout agitation. The bacterial suspensions were then centrifuged,washed, and re-suspended in the same volume of PBS and kept at 4° C.until use. In both inactivation methods, 0.1 ml of each suspension wasspread onto blood agar and incubated at 37° C. for up to one week tocheck for lack of growth. Before being used for oral immunization, thelevel of CS6 on the inactivated bacteria was checked by inhibitionELISA.

Mouse Immunizations and Sample Collection.

Groups of female Balb/C and C57 Bl/6 mice (Charles River; 6-8 weeks ofage; 5 mice/group) were used for oral (intragastric) immunizations. Allmice were given two doses of 3×10⁸ phenol-killed bacteria (inactivated,using 0.8% concentration of phenol) of either C600-CS6 or the referencestrain together with 7.5 μg CT two days apart in 0.3 ml 3% sodiumbicarbonate solution intragastrically through a baby feeding catheter(first round of immunization), followed two weeks later by two identicalimmunizations two days apart in a second round of immunization.Bleedings were performed before the first immunization and two weeksafter the last immunization, at which times fecal pellets (FPs) werealso collected and extracts prepared as described previously (Nygren E,Holmgren J, Attridge S R. Murine antibody responses following systemicor mucosal immunization with viable or inactivated Vibrio cholerae.Vaccine 2008; 26:6784-90). In addition, at the later time point when themice were sacrificed, they were perfused with a heparin-PBS solution toremove blood from the tissues, and small intestinal tissue collected andextracted with a 2% (w/v) Saponin-PBS solution (the Perfext method) asdescribed previously Villavedra M, Carol H, Hjulstrom M, Holmgren J,Czerkinsky C. “PERFEXT”: a direct method for quantitative assessment ofcytokine production in vivo at the local level. Res Immunol 1997;148:257-66).

ELISAs.

IgG+IgM and IgA antibody titers against CS6 were determined in sera,fecal and intestinal extracts, by ELISA, as described previously (RudinA, Svennerholm A-M. Colonization factor antigens (CFAs) ofenterotoxigenic Escherichia coli can prime and boost immune responsesagainst heterologous CFAs. Microb Pathog 1994; 16:131-9). CS6, for useas coating antigen (at the final concentration of 0.7 μg/ml) in ELISAs,was purified from the previously described TOP10-056 over-expressingstrain (Tobias J, Lebens M, Källgård S, Nicklasson M, Svennerholm A-M.Vaccine 2008; 26:5373-80), by sequential ammonium sulphate precipitationand gel filtration. Sera from individual mice were tested usinglow-binding microtiter plates (Greiner), and samples were initiallydiluted 1/100 followed by serial three-fold dilutions. Fecal pelletextracts and small intestine tissue extracts were tested in high-bindingmicrotiter plates (Greiner) in 3-fold serial dilutions from a startingdilution of 1/3. Antibody titers were calculated as the reciprocals ofthe sample dilutions which gave an A450 absorbance of 0.4 above thebackground value. In the fecal and intestinal extract samples, total IgAwas also measured by ELISA as described (Nygren E, Holmgren J, AttridgeS R. Vaccine 2008; 26:6784-90), and antigen-specific IgA antibody valueswere expressed as IgA titer units per μg of total IgA.

Statistical Analysis.

All ELISA experiments were done at least twice on different occasions.Statistical analyses were conducted by Student's t-test, and P<0.05(two-tailed) was regarded as a significant difference.

What is claimed is:
 1. A vaccine for immunization against coli surfaceantigen 6 “CS6”-expressing enterotoxigenic Escherichia coli “ETEC”,comprising a phenol-treated cell having an amount of CS6 antigenpresented on a cell surface that is at least 100% greater than anuntreated cell, wherein said 100% greater CS6 antigen presentationcorresponds to at least 0.6 μg/10⁹ bacteria.
 2. The vaccine according toclaim 1, wherein the amount of said antigen on the cell surface is atleast 200% greater than an untreated cell and corresponds to at least1.2 μg/10⁹ bacteria.
 3. The vaccine according to claim 2, wherein theamount of said antigen on the cell surface is at least 300% greater thanan untreated cell and corresponds to at least 1.8 μg/10⁹ bacteria. 4.The vaccine according to claim 1, wherein the cell was treated with 0.6to 2.0 percent phenol by weight for a duration of 1 to 72 h.
 5. Thevaccine according to claim 4, wherein the cell was treated for aduration of 1.5 to 42 h.
 6. The vaccine according to claim 1, whereinthe cell was treated with 0.6 to 2.0 percent phenol by weight at atemperature of 18 to 42° C.
 7. The vaccine according to claim 3, whereinthe cell was treated at a temperature of 18 to 42° C.
 8. The vaccineaccording to claim 7, wherein the cell was treated at a temperature of18 to 38° C.
 9. The vaccine according to claim 8, wherein the cell wastreated at a temperature of 18 to 22° C.
 10. The vaccine according toclaim 8, wherein the cell was treated at a temperature of 36 to 38° C.11. The vaccine according to claim 6, wherein the cell was treated witha phenol concentration of 0.8-2.0 percent by weight.
 12. The vaccineaccording to claim 11, wherein the cell was treated with a phenolconcentration of 1.0-2.0 percent by weight.
 13. The vaccine according toclaim 6, wherein the cell was treated with a phenol concentration of0.6-2.0 percent by weight, for a time of 6-72 h and at temperature of18-22° C.
 14. The vaccine according to claim 13, wherein the cell wastreated at a phenol concentration of 0.75-0.85 percent by weight, for atime of 40±2 h and at a temperature of 20±1° C.
 15. The vaccineaccording to claim 1, wherein the CS6 antigen is recombinantlyoverexpressed by the cell.
 16. The vaccine according to claim 1, whereinthe cell is a killed whole cell.
 17. The vaccine according to claim 16,wherein the cell is an Escherichia coli cell.
 18. A vaccine forimmunization against CS6-expressing ETEC, comprising a phenol treatedcell having an amount of ETEC CS6 antigen presented on a cell surfacegreater than an untreated cell, wherein the amount of surface CS6antigen presented is at least 0.6 μg/10⁹ bacteria, and wherein the cellwas treated with 0.6 to 2.0% phenol by weight for a duration of 1 to 72hours at a temperature of 18 to 42° C.
 19. The vaccine comprising aphenol treated cell according to claim 18, wherein the cell was treatedat a temperature of 20±1° C. with: 0.6% phenol by weight for a durationof 40 hours; 0.8% phenol by weight for a duration of 16 hours; 0.8%phenol by weight for a duration of 40 hours; 1.0% phenol by weight for aduration of 6 hours; 1.0% phenol by weight for a duration of 16 hours;1.0% phenol by weight for a duration of 40 hours; 1.2% phenol by weightfor a duration of 6 hours; 1.2% phenol by weight for a duration of 16hours; 1.2% phenol by weight for a duration of 40 hours; 1.5% phenol byweight for a duration of 6 hours; 1.5% phenol by weight for a durationof 16 hours; 1.5% phenol by weight for a duration of 40 hours; 2.0%phenol by weight for a duration of 6 hours; or 2.0% phenol by weight fora duration of 16 hours.
 20. The vaccine comprising a phenol treated cellaccording to claim 18, wherein the cell was treated with 1.0 to 1.5%phenol by weight for a duration of 6 to 40 hours at a temperature of20±1° C.
 21. A method of immunizing a subject against CS6antigen-expressing ETEC, the method comprising orally administering tothe subject an effective amount of a vaccine according to claim
 1. 22.The method according to claim 21, wherein the method comprises at leasttwo rounds of immunization separated by at least 2 days.
 23. A method ofimmunizing a subject against CS6 antigen-expressing ETEC, the methodcomprising orally administering to the subject an effective amount of avaccine according to claim
 18. 24. The method according to claim 23,wherein the method comprises at least two rounds of immunizationseparated by at least 2 days.